This invention relates to water-soluble fluorescent labeling reagents for biological investigations. Fluorescence detection techniques are dependent on the physical characteristics of the dyes that they employ. Many dyes that have excellent photophysical properties (high extinction coefficients and high quantum yields) have limited utility because other properties such as water solubility and photostability are poor. In particular, for most biological applications good aqueous solubility for the fluorophore is crucial.
A common problem with many commercially available fluorescent labeling reagents is that they are not made water soluble thus they must be dissolved in organic solvents, such as DMF, prior to substrate labeling in aqueous media. Such organic solvents can have a deleterious effect upon sensitive substrates. The solubility of organic dyes affects the degree to which they interact with themselves in solution and when conjugated to substrates and therefore directly influences their light absorption and emission properties. The problem of non specific staining of cellular matter by the dye, which reduces signal to noise during observation, is also a function of the dye's hydrophobicity and of the polarity of its appended functional groups. In addition, the dye should not cause precipitation of the substrate once labeling has occurred. There is a need for fluorescent reagents that are bright, that have good aqueous solubility, and that give low non specific staining.
In recent years, a series of reagents based sulphocyanine dyes has become commercially available; see U.S. Pat. Nos. 5,268,486 and 5,486,616 the disclosures of which are hereby incorporated by reference. These dyes, which are highly polar due to the presence of two sulphonic acid residues, have excellent photophysical properties combined with good aqueous solubility. To determine if further improvements could be achieved for the properties of cyanine dye based fluorescent reagents, the sulphonic acid groups were replaced with carbohydrate residues to create water soluble cyanine dyes with reduced polarity and labeling capabilities for biological and non-biological substrates.